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| Cloning, Subcellular Localization and Functional Analysis of GlbHLH1 Gene in Ganoderma lingzhi |
| WANG Yi-Yi1,2, XU Juan1,2,3, RUAN Shi-Yu1,2, ZHANG Yi1, XING Bing-Cong1,2, CHEN Bin-Huang1, WU Xue-Qian1,2,3,* |
1 College of Food and Health, Zhejiang A&F University, Lin'an 311300, China;
2 Zhejiang Provincial Key Laboratory of Characteristic Traditional Chinese Medicine Resources Protection and Innovative Utilization, Lin'an 311300, China;
3 National Innovation Alliance of Lingzhi Sanye Green Industry, Lin'an 311300, China |
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Abstract Methyl jasmonate (MeJA) can induce the biosynthesis of Ganoderma terpenes, in order to explore the relationship between Ganoderma terpene biosynthesis and the regulation of basic helix-loop-helix (bHLH) genes and jasmonic acid (JA) signal regulation. In this study, the bHLH gene fragment related to the biosynthesis of Ganoderma terpenoids was cloned from G. lingzhi by homologous alignment. The full length of the gene was further cloned and analyzed its bioinformatics, transcriptional activity, prokaryotic expression, subcellular localization, overexpression and silencing expression. The results showed that a bHLH gene fragment with high homology with the positively regulated tanshinone synthesis transcription factor was obtained from G. lingzhi. qRT-PCR analysis showed that the gene was significantly indigenous in response to MeJA induction. The expression levels of the 3 key genes in the triterpenoid (GA) synthesis pathway of G. lingzhi were significantly increased under MeJA induction (P<0.05), and the expression pattern was consistented with bHLH. A 1 311 bp GlbHLH1 gene (GenBank No. MW981280.1) was cloned, encoding 436 amino acids with a molecular weight of 46.33 kD. Promoter analysis showed that G-box binding to bHLH transcription factor existed in the promoter regions of the 3 key genes in Ganoderma triterpene synthesis pathway, suggested that GlbHLH1 might be involved in the regulation of Ganoderma triterpene metabolism by responding to JA signal. Phylogenetic analysis showed that GlbHLH1 had the closest relationship with GsPIL37177.1 of G.sinense; the transcriptional activation activity in Saccharomyces cerevisiae showed that the gene had transcriptional activation activity. The prokaryotic expression vector of GlbHLH1 gene was successfully constructed, and the fusion protein of expected size (about 75 kD) was expressed in Escherichia coli BL21. Subcellular localization showed that GlbHLH1 was mainly located in the nucleus; the transgenic results showed that the expressions of GlbHLH1 in the overexpression and silencing strains were significantly up- and down-regulated from that in the wild type (P<0.05), the triterpenoid content in the overexpression strains OE-GlbHLH1-1, OE-GlbHLH1-2 and OE-GlbHLH1-3 increased by 25%, 22% and 38%, respectively, and that in the silencing strains Si-GlbHLH1-1 and Si-GlbHLH1-2 was decreased by about 15% and 30%, respectively. In summary, GlbHLH1 is an important transcription factor that controls the synthesis of triterpenoids in G. lingzhi. The above results provide a theoretical basis for further study on the molecular regulation mechanism of bHLH in G. lingzhi.
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Received: 13 February 2022
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Corresponding Authors:
* wxq@zafu.edu.cn
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[1] 崔小清, 丁壮, 武莉, 等. 2017. 生合成途径特异性转录因子及其激活真菌沉默次级代谢产物的研究进展[J]. 中国海洋药物, 36(04): 75-82.
(Cui X Q, Ding Z, Wu L, et al.2017. Research progress on the pathway-specific transcription factors and their application in activating cryptic fugal secondary metabolites[J] Chinese Journal of Marine Drugs, 36(04): 75-82.)
[2] 戴玉成, 曹云, 周丽伟, 等. 2013. 中国灵芝学名之管见[J]. 菌物学报, 32(06): 947-952.
(Dai Y C, Cao Y, Zhou L W, et al.2013. Notes on the nomenclature of the most widely cultivated Ganoderma species in China[J]. Mycosystema, 32(06): 947-952.)
[3] 戴玉成, 杨祝良, 崔宝凯, 等. 2021. 中国森林大型真菌重要类群多样性和系统学研究[J]. 菌物学报. 40(04): 770-805.
(Dai Y C, Yang Z L, Cui B K, et al.Diversity and systematics of the important macrofungi in Chinese forests[J]. Mycosystema, 40(04): 770-805.)
[4] 高珂, 王玲, 吴素瑞, 等. 2015. 调控药用植物药效成分合成的转录因子研究进展[J]. 中草药, 46(20): 3100-3108.
(Gao K, Wang L, Wu S R, et al.2015. Advances in studies on transcriptional factor regulation of biosynthesis of active components in medicinal plants[J]. Chinese Traditional and Herbal Drugs, 46(20): 3100-3108.)
[5] 朱静, 宋书琪, 岳思宁, 等. 2020. 灵芝转录因子AreA的生物学功能[J]. 菌物学报, 39(01): 57-65.
(Zhu J, Song S Q, Yue S N, et al.2020. Biological function of the transcription factor AreA in Ganoderma lingzhi[J]. Mycosystema, 39(01): 57-65.)
[6] Abdullah S, Oh Y S, Kwak M K, et al.2021. Biophysical characterization of antibacterial compounds derived from pathogenic fungi Ganoderma boninense[J]. Journal of Microbiology, 59(2): 164-174.
[7] Chen S L, Xu J, Liu C, et al.2012. Genome sequence of the model medicinal mushroom Ganoderma lucidum[J]. Nature Communications, 3: 913.
[8] Du T Z, Niu J F, Su J, et al.2018. SmbHLH37 functions antagonistically with SmMYC2 in regulating jasmonate-mediated biosynthesis of phenolic acids in Salvia miltiorrhiza[J]. Frontiers in Plant Science, 9: 1720.
[9] Gong T, Yan R Y, Kang J, et al.2019. Chemical components of Ganoderma[J]. Advances in Experimental Medicine and Biology, 1181: 59-106.
[10] Hong G J, Xue X Y, Mao Y B, et al.2012. Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression[J]. Plant Cell, 24(6): 2635-2648.
[11] Ji Y P, Xiao J W, Shen Y L, et al.2014. Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua[J]. Plant & Cell Physiology, 55(9): 1592-1604.
[12] Jiang A L, Liu Y N, Liu R, et al.2019. Integrated proteomics and metabolomics analysis provides insights into ganoderic acid biosynthesis in response to methyl jasmonate in Ganoderma lucidum[J]. International Journal of Molecular Sciences, 20(24): 6116.
[13] Jin J P, Tian F, Yang D C, et al.2017. PlantTFDB 4.0: Toward a central hub for transcription factors and regulatory interactions in plants[J]. Nucleic Acids Research, 45(D1): D1040-D1045.
[14] Keller N P.2019. Fungal secondary metabolism: Regulation, function and drug discovery[J]. Nature Reviews Microbiology, 17(3): 167-180.
[15] Kladar N V, Gavaric N S, Bozin B N.2016. Ganoderma: Insights into anticancer effects[J]. European Journal of Cancer Prevention, 25(5): 462-471.
[16] Liu C D, Dunkin D, Lai J, et al.2015. Anti-inflammatory effects of Ganoderma lucidum triterpenoid in human crohn's disease associated with downregulation of NF-kappaB signaling[J]. Inflammatory Bowel Diseases, 21(8): 1918-1925.
[17] Mertens J, Pollier J, Vanden B R, et al.2016. The bHLH transcription factors TSAR1 and TSAR2 regulate triterpene saponin biosynthesis in Medicago truncatula[J]. Plant Physiology, 170(1): 194-210.
[18] Pan H T, Wang Y J, Na K, et al.2019. Autophagic flux disruption contributes to Ganoderma lucidum polysaccharide-induced apoptosis in human colorectal cancer cells via MAPK/ERK activation[J]. Cell Death & Disease, 10(6): 456.
[19] Patra B, Pattanaik S, Schluttenhofer C, et al.2018. A network of jasmonate-responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. New Phytologist, 217(4): 1566-1581.
[20] Peng J J, Wu Y C, Wang S Q, et al.2020. SmbHLH53 is relevant to jasmonate signaling and plays dual roles in regulating the genes for enzymes in the pathway for salvianolic acid B biosynthesis in Salvia miltiorrhiza[J]. Gene, 756: 144920.
[21] Pierre B.2004. Transcription factors as tools for metabolic engineering in plants[J]. Current Opinion in Plant Biology, 7(2): 202-209.
[22] Sharma P, Tulsawani R.2020. Ganoderma lucidum aqueous extract prevents hypobaric hypoxia induced memory deficit by modulating neurotransmission, neuroplasticity and maintaining redox homeostasis[J]. Scientific Reports, 10(1): 8944.
[23] Shen Q, Lu X, Yan T X, et al.2016. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua[J]. New Phytologist, 210(4): 1269-1281.
[24] Suwannarach N, Kumla J, Sujarit K, et al.2020. Natural bioactive compounds from fungi as potential candidates for protease inhibitors and immunomodulators to apply for Coronaviruses[J]. Molecules, 25(8): 1800.
[25] Van Moerkercke A, Steensma P, Schweizer F, et al.2015. The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus[J]. Proceedings of the National Academy Sciences of the USA, 112(26): 8130-8135.
[26] Wu F, Zhou L W, Yang Z L, et al.2019. Resource diversity of Chinese macrofungi: Edible, medicinal and poisonous species[J]. Fungal Diversity, 98(1): 1-76.
[27] Xing B C, Liang L J, Liu L L, et al.2018. Overexpression of SmbHLH148 induced biosynthesis of tanshinones as well as phenolic acids in Salvia miltiorrhiza hairy roots[J]. Plant Cell Reports, 37(12): 1681-1692.
[28] Xing B C, Yang D F, Yu H Z, et al.2018. Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots[J]. Plant Science, 276: 229-238.
[29] Zhang Q Z, Wu N Y, Jian D Q, et al.2019. Overexpression of AaPIF3 promotes artemisinin production in Artemisia annua[J]. Industrial Crops and Products, 138: 111476.
[30] Zhang X, Luo H M, Xu Z C, et al.2015. Genome-wide characterisation and analysis of bHLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza[J]. Scientific Reports, 5: 11244.
[31] Zhou Y Y, Sun W, Chen J F, et al.2020. SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza[J]. Scientific Reports, 10(1): 7201. |
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